1. Field of the Invention
The present invention relates to a method and apparatus for discriminating (sorting or selecting) minute particles such as organic polymers or biological particles, e.g., polymers in cells. More particularly, the present invention relates to a method and apparatus wherein a light pulse of a high-intensity light beam such as a laser beam is irradiated onto a continuous flow of minute particles in suspension to cause the minute particles to emit fluorescence or phosphorescence; a change in the intensity of emitted light over a period of time is automatically measured and analyzed to discriminate the minute particles; and the minute particles are physically sorted in accordance with the discrimination result.
2. Description of the Prior Art
In cytology, there is an increasing demand for automatic cell analysis and sorting. Presently, the screening of cytological materials to detect, for example, cancerous or malignant cells, is typically performed using a hierarchy of two or more levels of screening. First, cell samples are visually prescreened by an observer to search out those that appear to contain abnormal cells. These samples are then later examined by a trained cytotechnologist or pathologist to determine whether the cells are indeed cancerous or malignant. Although this method works well, it is also subject to a number of disadvantages, i.e., it is time-consuming and requires a trained technician, thereby making the method costly. Furthermore, the method is nonquantitative, since the criteria of abnormality are largely subjective. Because of the time and cost, it is difficult to apply this method to very large cell populations.
For the analysis of biological minute particles, flow system analysis is called flow cytometery. This method has the potential to provide some solution to the above problems. In flow cytometry, an optical or electrical signal is obtained which represents information on cell suspension, and cell properties or structures are analyzed in accordance with such a signal. In a flow cytometer based on flow cytometry, light is irradiated onto a cell suspension flowing at high speed in a very small detection volume, and an optical signal or electrical signal is obtained from information returned from the cell. Flow cytometry thus allows the discrimination of normal and abnormal cells within a short period of time, and provides quick automatic cell screening.
A continuous laser beam is used as the irradiating light. However, a single parameter such as cell size is frequently insufficient to allow correct cell analysis. In view of this, multiparameter analysis involving light absorption, fluorescence, scatter, and the like is presently performed. Cell size is used as a parameter when cells of a particular type have different sizes in normal and abnormal states. Thus, normal cells can be differentiated from abnormal cells by observing the cell size. When a particular type of cell does not allow discrimination between normal and abnormal cells, using only the cell size as a parameter, an additional parameter which does allow such discrimination is used. Thus, multiparameter analysis increases the ability to distinguish differences between cells of a particular type.
A cell sorter not only sorts minute particles in accordance with optical or electrical signals representing particular information on cells, but also performs cell grouping based on signal processing, such as grouping into normal and abnormal cells.
A flow cytometer and cell sorter are reported by Noguchi in Denshi Gijutsu Sogo Kenkyusho Iho, Vol. 44, No. 3 (1980). A cell sorter is also described in Japanese Examined Patent Publication (Kokoku) Nos. 46-27118 and 56-13266.
Unlike cell analysis under microscopic observation or by means of cell samples, in flow system analysis using a flow cytometer a large number of cells are allowed to flow and can be discriminated within a short period of time. This method allows statistical processing of the cells. However, a parameter has not been available that allows discrimination based on highly significant information directly related to the abnormality of cells, such as information on the cell morphology, molecular biological information, or molecular chemical information. Since such highly significant information is directly related to the occurrence of abnormalities in cells, its use improves the screening precision due to an increase in the number of parameters. This contributes greatly to clinical applications and fundamental medical studies.
The present inventors have studied the possibility of adding a new parameter to the conventional flow system analysis method that allows the discrimination of cells based on such highly significant information.
Meanwhile, in the field of laser technology, a technique of a short pulse train tunable laser is being established. With the recent advent of a dye laser or the like using a dye solution as a laser medium, a laser beam tunable to any wavelength within a wide range from ultraviolet to infrared rays can be obtained. The tunable laser can provide a light pulse of a very short duration.
The mode locking method is known as a method of obtaining such a short light pulse. Although various methods of achieving mode locking are known, the synchronous locking method is most frequently used. According to the synchronous locking method, a short light pulse from a mode-locked argon ion laser is used as an excitation light source for exciting a dye laser; the resonance period of a resonator in the dye laser is set to coincide with the period of the obtained pulse train; and the gain of the dye laser is periodically modulated to generate an ultra-short light pulse train.
When this mode locking method is used, a tunable light pulse train is obtained which has a pulsewidth of several nanoseconds (10.sup.-9 seconds) to several picoseconds (10.sup.-12 seconds) and has a high output, monochromatic property, and directivity. The period of the pulse train can be kept variable within a range of several milliseconds to several nanoseconds by the cavity damping method.
An output laser beam obtained by such a method generally comprises linearly polarized light, since an optical element used in the laser resonator is set at the Brewster angle and the excited laser beam is linearly polarized light.
When such light changing at high speed is irradiated onto a substance, to cause this substance to emit light, changes in the intensity of emitted light over a period of time also occur at high speed. This means that the observation or measurement of emitted light must be performed with high precision with respect to time.
When the observation unit time is longer than a subnanosecond, a change in the intensity of emitted light is converted into an electrical signal by a photosensor, such as a photodiode or a photoelectron multiplier, and the obtained electrical signal is processed as needed. The processed signal is supplied to an oscilloscope, for example, to display an image on a CRT monitor, or is counted by a photon counting method.
In the measurement of ultra high-speed time units such as a picosecond or subpicosecond, a photosensor as described above presents the problems of response speed and sensitivity. Therefore, a streak camera is used in this case. In order to measure slight, repetitive, and ultra high-speed changes in light intensity, a synchro scan streak camera can be conveniently used. In a streak camera, photoelectrons from a surface which receives emitted light are accelerated and deflected in such a manner that a change in the intensity of the received light is translated into a change in geometrical position of an image on a CRT monitor.
Attempts are being made to use a photochemically reactive substance such as a hemoglobin complex to measure a change in the intensity of transient emitted light over a period of time upon short pulse train laser excitation, as described above. The application of such a method to the analysis of biological polymers is also proposed, and is expected to be effective in the analysis of the local structure of such biological polymers. However, these subjects are still in the state of preliminary studies with cell samples.
In view of this situation, the present inventors have made studies to determine the plausibility of combining this method with flow system analysis, in order to allow on-line selecting and/or sorting of minute particles in accordance with a different in the local structure at the molecular level of minute particles such as biological particles or organic polymers.